Electrochemical, particularly amperometric, chronoamperometric, and chronocoulometric sensors of hydrogen peroxide are used in the assay of biochemicals including glucose, lactate, and cholesterol, as well as in the detection and assay of immunoreagents, in nucleic acid sensors, and in sensors detecting other affinity reactions. Hydrogen peroxide biosensors are also used to monitor biochemicals in vivo, such as glucose and lactate. In vivo monitoring of glucose is relevant to the management of diabetes mellitus. Monitoring of lactate is important to the confirmation of hypoxia or ischemia.
Glucose can be monitored subcutaneously or transcutaneously, i.e., by transporting glucose across the skin and measuring its concentration. A general method for assaying glucose involves reacting glucose in a test sample with molecular oxygen in the presence of excess glucose oxidase. The reaction produces hydrogen peroxide and gluconolactone, and the hydrogen peroxide is sensed with a hydrogen peroxide sensor. For measuring lactate or cholesterol, hydrogen peroxide is similarly produced in the respective presence of lactate oxidase or cholesterol oxidase.
Hydrogen peroxide is electrochemically detected by its electrooxidation on a platinum or other relatively inert platinum-group metal electrode. Platinum electrodes, however, become fouled in biological fluids, limiting their use. Unless the surface of the platinum electrode is cleaned or reconditioned periodically, e.g., by chemical cleaning or electrochemical oxidation and reduction, the potential dependence of the rate of electrooxidation of hydrogen peroxide, and thus the sensitivity of the platinum-based detector, changes.
The fouling problem was previously addressed through use of non-corroding metal or carbon electrodes coated with films comprising peroxidase, e.g., horseradish peroxidase. In such sensors, the peroxidase was oxidized by hydrogen peroxide, and then electroreduced either directly or through a process involving a diffusing electron-relaying species.
Amperometric enzyme electrodes containing peroxidase for hydrogen peroxide detection have been described. In these prior art sensors, peroxidase is obtained from horseradish, Arthromyces ramosus, and bovine milk. See, for example, Kulys and Schmid, 1990, Bioelectrodchem. Bioenerg. 24:305-311; Wang et al., 1991, Anal. Chim. Acta, 254:81-88; and Gorton et al., 1991, Anal. Chim. Acta 250:203-248; and Paddock et. al., 1989, J. Electroanal. Chem. Interfacial Electrochem. 260:487-494. However, these sensors could not be operated for prolonged periods of time at 37.degree. C. or above without significant loss of sensitivity.
Prior art peroxidase electrodes also included those based on "wiring" enzymes to electrodes through electron-conducting hydrogels. Because the enzyme "wired" hydrogels are highly permeable to hydrogen peroxide and conduct electrons, covalent bonding of the peroxidase to the cross-linked polymer network yielded hydrogen peroxide sensors with high sensitivity. See, for example, Gregg and Heller, U.S. Pat. No. 5,320,725; Vreeke and Heller, 1994, In: Diagnostic Biosensor Polymers; Usmani and Akmal, eds., ACS Symposium Series 556; and Vreeke et al., 1992, Anal. Chem. 64:3084-3090 which are hereby incorporated by reference. The current of these "wired" peroxidase sensors is mass transport controlled over a broad concentration range and thus varies linearly with the hydrogen peroxide concentration. In the "wired" peroxidase sensors, the peroxidase is covalently bound to a redox polymer network of an electron-conducting hydrogel coating the electrode. The sensitivity of these sensors is as high as 1 Acm.sup.-2 M.sup.-1. These sensors also have less noise than platinum electrodes at equal current density, and, unlike platinum electrodes, they are not fouled in biological solutions.
Although the operational life of the "wired" peroxidase sensor is sufficiently long to be adequate for most applications at 25.degree. C., its operational life is severely reduced at temperatures at or above 37.degree. C. For example, when used continuously for several days at or near the temperature of the human body, 37.degree. C., the wired peroxidase sensor lost its sensitivity. In addition, because the sensor rapidly lost its sensitivity at temperatures above 37.degree. C., it could not be used for prolonged periods in bioreactors or bioanalytical systems operating at or above 37.degree. C. Furthermore, nucleic acid-sensing by electrochemical processes which rely on denaturing paired nucleic acid strands at temperatures in excess of 50.degree. C. requires thermostable electrochemical devices.
Many attempts have been made to improve the operational stability of enzyme biosensors. Enzyme electrodes designed with an excess of enzyme and a membrane to reduce the flux of substrate have been used to improve the operational stability of enzyme electrodes. See, for example, the Instruction Manual for YSI Model 23A and 23 AM glucose analyzer produced by Yellow Springs Instruments, Inc., Yellow Springs, Calif., and Alvarez-Icaza and Bilitewski, 1993, Anal. Chem. 65:525A-533A. These sensors maintain their performance specifications even after most of their enzyme has become inactive, because residual enzyme suffices to completely convert the restricted substrate flux.
Other stabilization methods focused on slowing the inactivation of the enzyme. Researchers who assumed that enzyme inactivation resulted from an irreversible change in protein folding, modified the protein surface or fixed its structure by cross-linking for better stability. See, for example, Chien et al., 1994, Biotechnol. Appl. Biochem; 19:51-60; Asther and Muenier, 1993, App. Biochem. Biotechnol. 38:57-67; and Mosbach, K., Ed., 1988, Methods in Enzymology, Academic Press, San Diego, Vol 135-137.
Thermostability was improved by covalently bonding enzyme in a phospholipid modified surface that resembled the enzyme's natural, membrane environment. See, for example, Kallury et al., 1992, Anal. Chem., 64:1062-1068 and 1993, 65:2459-2467. However, the lipids and the substrates to which the lipids were bound were insulating. Therefore these lipid-stabilized systems could not be useful as thermostable electrochemical sensors. Enhancement of sensor thermostability using enzymes isolated from thermophilic organisms has been considered theoretically, but these enzymes are unavailable in the needed quantity and/or do not have the required activity. Thermostable biosensors remain commercially unavailable. Moreover, those thermostable enzymes that were proposed for use in biosensors were not enzymes that could be used in the assay of hydrogen peroxide.
Soybean peroxidase has recently become commercially available. While the manufacturer claims that the enzyme is stable at 80.degree. C. for 12 hours, its stability and sensitivity in electrochemical reactions are unknown. The enzyme has been disclosed as useful in biochemical and immunological assays in which horseradish peroxidase is useful (U.S. Pat. No. 5,278,046). The utility of this enzyme in electrochemical applications is unknown.
Thus, despite advances in operational stability of enzyme electrodes, stable, sensitive electrodes for the detection of hydrogen peroxide are not available. It would be highly desirable to provide a stable and sensitive biosensor for the detection of hydrogen peroxide which is thermostable, e.g., able to withstand operation at temperatures of 37.degree. C. and higher for sustained periods of time.